Immunosuppressive agents

ABSTRACT

A compound with immunosuppressive properties having formula (I), in which R 1  represents a heterocyclic ring system, a combined heterocyclic ring system and homocyclic ring system, or an ethoxycarbonylmethyl group. R 2  and R 3  represent hydrogen or lower alkyl groups.

This is a continuation of Application Ser. No. 07/915,784 filed on Jul.16, 1992, which is a continuation of application Ser. No. 07/663,873filed Mar. 6, 1991 both abandoned.

This invention relates to immunosuppressive agents.

The purine derivative azathioprine interfers with cell replication, andhas immunosuppressive and antileukamic properties. It is usedextensively as a drug to treat a wide range of chronic inflammatorydiseases in which immune mechanisms are involved such as rheumatoidarthritis; chronic active hepatitis; kidney disease; skin disease; andmultiple sclerosis. It is also used to inhibit the rejection oftransplanted organs such as kidneys. However, its beneficial therapeuticeffects are compromised by the fact that it is toxic to bone marrow. Itis the object of the present invention to provide an immunosuppressiveagent which does not have this disadvantage.

According to one aspect of the invention there is provided a compoundfor use as an immunosuppressive agent, said compound having the formula:##STR1## in which R¹ comprises a heterocyclic ring system, a homocyclicring system, a combined heterocyclic and homocyclic ring system,alkoxycarbonylalkyl or aryloxycarbonylalkyl group and wherein

R¹ does not comprise ##STR2## or isomers thereof. R¹ is alkyl, aryl orhydrogen and

R³ is hydrogen, alkyl, aryl or nitroaryl Preferably

R² is hydrogen, methyl, ethyl, or propyl; and

R³ is hydrogen, methyl or 4-nitrophenyl.

According to a preferred embodiment of the invention said compoundswhich are used as immunosupressive agents have the following formula:##STR3## in which R² is hydrogen, or methyl or ethyl or propyl; and

R³ is hydrogen or methyl or 4-nitrophenyl, and in which R¹ may be one ofthe following: ##STR4## in which R⁴ is hydrogen or methyl or ethyl orpropyl or phenyl or 1-naphthyl 2. ##STR5## in which R⁵ is hydrogen ortrifluoromethyl or phenyl or 4-chlorophenyl or 4-methoxyphenyl or3-pyridyl 3. ##STR6## in which R⁶ hydrogen or phenyl or chlorophenyl or4-methoxyphenyl 4. ##STR7## in which R¹⁰ is hydrogen or an alkoxy group;and

R⁷ is hydrogen, alkyl, nitro, halide or an alkoxy group; and

R⁸ is hydrogen, halide or an alkoxy group; and

R⁹ is hydrogen or an alkoxy group 5. ##STR8## in which R¹¹ is hydrogen,methyl, or hydroxyl; and

R¹² is hydrogen or methyl; and

X is nitrogen or C--H 6. ##STR9## in which Z is oxygen or sulphur orN--H; and

Y is C--H or nitrogen 7. ##STR10## in which R¹³ is hydrogen or an aminoor carboxyl group; and

R¹⁴ is hydrogen or methyl; and

R¹⁵ is hydrogen or methyl 8. ##STR11## in which R¹⁶ is alkyl or aryl 9.##STR12## 10. in which

R¹⁷ is alkyl or aryl 11. ##STR13## 12. in which R¹⁸ is alkyl or aryl 13.##STR14##

Specific illustrative examples of the first embodiment of the inventionare as follows: ##STR15##

Some of the compounds, as referred to above are previously unknown.

Therefore, according to a further aspect of the invention there areprovided compounds comprising the following formula: ##STR16## whereinthe molecular groups R¹, R², and R³ are defined as one of the following:

1. R¹ is defined as the following: ##STR17## in which R⁴ is hydrogen ormethyl;

R² is ethyl; and

R³ is methyl OR

R² is methyl and R³ is hydrogen; and

R⁴ is phenyl or 1-naphthyl OR

2. R¹ is defined as the following: ##STR18## in which R²,R³, and R⁵ areone of the following combinations:

R² is methyl, R³ is 4-nitrophenyl, and R⁵ is hydrogen; or

R² is methyl, R³ is hydrogen, and R⁵ is trifluoromethyl; or

R² is ethyl, R³ is methyl, and R⁵ is hydrogen; or

R² is methyl, R³ is hydrogen, and R⁵ is phenyl; or

R² is methyl, R³ is hydrogen, and R⁵ is 4-chlorophenyl; or

R² is methyl, R³ is hydrogen, and R⁵ is 4-methocyphenyl; or

R² is methyl, R³ is hydrogen, and R⁵ is 3-pyridyl OR

3. R¹ is defined as the following: ##STR19## in which R² is methyl; and

R³ is hydrogen; and

R⁶ is hydrogen or phenyl or 4-chlorophenyl o 4-methoxyphenyl OR

4. R¹ is defined as the following: ##STR20## in which R² is methyl; and

R³ is hydrogen, and

R⁷, R⁸, R⁹, R¹⁰ are one of the following combinations:

R⁸, R⁹, R¹⁰ are all hydrogen and R⁷ is methyl; or

R⁸, R⁹, R¹⁰ are all hydrogen and R⁷ is a nitro group; or

R⁹ and R¹⁰ are both hydrogen, R⁷ is fluorine, and R⁸ is chlorine; or

R⁹ and R¹⁰ are both hydrogen, and R⁷ and R⁸ are both methoxy groups; or

R⁷ and R⁸ are both hydrogen and R⁹ and R¹⁰ are both methoxy groups OR

5. R¹ is defined as the following: ##STR21## in which R² is methyl; and

R³ is hydrogen; and

R¹¹,R¹² and X are defined as one of the following combinations:

R¹¹ and R¹² are both hydrogen, and X is C--H; or

R¹¹ is a hydroxy group, R¹² is hydrogen, and X is nitrogen OR

R² is ethyl; and

R³, R¹¹ and R¹² are all methyl; and

X is nitrogen OR

6. R¹ is defined as the following: ##STR22## in which R² is methyl; and

R³ is hydrogen; and

Y and Z are defined as one of the following combinations:

Y is C--H and Z is oxygen; or

Y is nitrogen and Z is sulphur; or

Y is nitrogen and Z is N--H OR

7. R¹ is defined as the following: ##STR23## in which R² is methyl; and

R³ is hydrogen; and

R¹³,R¹⁴, and R¹⁵ are defined in either of the following combinations:

R¹³ is hydrogen, and R¹⁴ and R¹⁵ are both methyl; or

R¹³ is carboxyl, and R¹⁴ and R¹⁵ are both hydrogen OR

8. R¹ is defined as the following: ##STR24## in which R² is methyl; and

R³ is hydrogen; and

R¹⁶ is methyl or phenyl OR

9. R¹ is defined as the following: ##STR25## OR

10. R¹ is defined as the following: ##STR26## OR

11. R¹ is defined as the following: ##STR27##

Referring now to Reaction Scheme 1, azathioprine I was originallydesigned as a "pro-drug" for 6-mercaptopurine II, to which it is rapidlyconverted by red blood cells. 6-mercaptopurine II has immunosupressiveproperties, but the mechanism of its action is unclear. ##STR28##

It would appear that the immunosuppressive action of azathioprine I isdue not solely to prior conversion of 6-mercaptopurine II in the body,but to the immunosuppressive action of azathioprine I itself. Themechanism of action of azathioprine I differs from that of6-mercaptopurine II. There is strong evidence to suggest that6-mercaptopurine II is converted in vivo into a nucleotide metabolitethat is associated with bone marrow toxicity. There is no evidence tosuggest that metabolites derived from the 1-methyl-4-nitroimidazolemoiety of azathioprine give rise to toxicity.

It is thought L-hat azathioprine I alkylates thiol groups in thelymphocyte cell membrane, most probably by a process ofaddition-elimination with the consequent release of 6-mercaotopurine II.It is further supposed that the alkylation of the lymphocyte results inimmunosuppression, whilst the 6-mercaptopurine II gives rise, as statedabove, via its nucleotide metabolite, to bone marrow toxicity. Supportfor this hypothesis is provided by the fact that lymphocyte cellmembranes are richly endowed with thiol groups, and by the knownpropensity of azathioprine to react with thiols both in vitro and invivo.

5-(1-alkyl-4-nitroimidazole) derivatives VI of relatively non toxicalkyl thiols or aryl thiols V are therefore suitable candidates forevaluation as good, relatively non toxic immunosuppressive agents, as 5shown in Reaction Scheme 2. ##STR29##

Examples of immunosuppressive agents of this kind are shown previouslyin examples 1-43. It is not intended that the invention be limited tothese illustrative examples.

Compounds 1 to 17, 19 to 22, 25 to 32, 34 and 36 to 43 may besynthesised, for example, by reaction of5-chloro-1-methyl-4-nitroimidazole 1V with the appropriate thiol V andpotassium carbonate in a suitable solvent such as, for example,tetrahydrofuran, dimethylformamide, or water. Compounds 18, 23, 24 and35 may be synthesised, for example from5-chloro-1-ethyl-2-methyl-4-nitroimidazole VII and the appropriate thiolunder suitable conditions, such as, for example, in acetone in thepresence of potassium carbonate.

Sixteen of these compounds were screened for immunosuppressive activityby means of the human mixed lymphocyte reaction, which is well known andneed not be further described here, the results of which are shown inTable 1.

                  TABLE 1                                                         ______________________________________                                                     Concentration                                                                              % Inhibition                                        Compound     of Solution (μM)                                                                        of .sup.3 M-Thymidine                               ______________________________________                                        I (azathioprine)                                                                           25           79                                                  21           10           89                                                  22           10           82                                                  32           25           98                                                   1           36           98                                                   2           25           86                                                   4           50           59                                                   5           25           94                                                   6           25           41                                                  15           25           76                                                  16           25           47                                                  19           25           29                                                  18           25           47                                                   8           25           87                                                   9           25           27                                                  13           25           34                                                  30           25           65                                                  ______________________________________                                    

The individual figures in the right hand column of the above table aredirectly proportioned to immunosuppressive activity.

As can be seen from Table 1 all of the compounds screened forimmunosuppressive activity displayed significant immunosuppression, andsix were substantially more active than azathioprine at the sameconcentration (25 μM).

Table 2 shows the measure of the immunosuppressive activity and toxicityof compounds 21,22,32, and 11.

                  TABLE 2                                                         ______________________________________                                                   Immunosuppressive      Therapeutic                                            activity.sup.a                                                                              Toxicity.sup.b                                                                         index                                       Compound   ED.sub.50     LD.sub.25                                                                              ED.sub.50 /LD.sub.25                        ______________________________________                                        I (azathioprine)                                                                         7.9           42.5     5.38                                        21         2.8           c        vh                                          22         3.15          d        vh                                          32         1.6                                                                11         24.0          e        vh                                          ______________________________________                                         .sup.a ED.sub.50 = concentration (μM) which brings about 50% inhibitio     of .sup.3 Hthymidine incorporation in the human mixed lymphocyte reaction     lower figures indicate increased reactivity                                   .sup.b LD.sub.25 = concentration (μM) at which 25% of the cells are        killed: lower figures indicate increased toxicity                             .sup.c No LD.sub.25 because only 19% of cells killed at 100 μM             .sup.d No LD.sub.25 because only 15% of cells killed at 215 μM             .sup.e No LD.sub.25 because only 16% of cells killed at 25 μM              (For c, d and e these are maximum vaules: no more cells were killed at        higher concentrations)                                                        vh very high                                                             

Referring now to Table 2, imidazole derivatives 21 and 22 and thetriazole derivative 32 are shown to be substantially moreimmunosuppressive than azathiourine and also considerably less toxic tolymphocytes. Therapeutic indices for these compounds are therefore atleast an order of magnitude greater than for azathioprine.

The compound 11 in which the 6-mercaptopurine moiety of azathioprine isreplaced by 8-hydroxy-6-mercaptopurine moiety, is less immunosuppressivethan azathioprine according to the mixed human lymphocyte reaction, asis shown in Table 2, but is much less toxic to the lymphocytes. Thetherapeutic index of compound is therefore much greater than that ofazathioprine, according to these tests. The significance of this resultresides in the fact that 8-hydroxy-6-mercaptopurine VIII is known to bea non toxic metabolite of azathioprine, and its metabolic fate is known.

Compound 32 showed pronounced immunosuppressive activity in vivo. It wastested in CBA mice transplanted with skin from Balb/c mice, according tothe skin grafting technique described by Billingham and Medawar (J.Exp.Biol, 28, 385-405 (1951). The effect compound 32 had on skin graftsurvival was compared with a control of saline, and with the standardimmunosuppressive agent azathioprine. All drugs were injected into theperitoneum starting three hours before surgery and then daily until thegraft had fully rejected. Mice were placed in groups at random, andrejection of the graft was evaluated by visual inspection by anindependent observer. The results are recorded in Table 3.

                  TABLE 3                                                         ______________________________________                                                  Number   Dosage*    Graft survival                                  Treatment of mice  (mg/Kg/day)                                                                              (Mean ± SEM days)                            ______________________________________                                        Control   28       NA         12.5 ± 0.4                                   Compound 32                                                                             24       45         14.2 ± 0.4                                   athloprine                                                                              29       52         12.4 ± 0.3                                   ______________________________________                                         *Compound 32 and azathioprine were administered in equimolar amounts     

The prolongation of graft survival caused by compound 32 (1.7 days)compared to a saline control is significant statistically (p=5.9×10⁻³).Similarly, the prolongation of graft survival caused by compound 32 (1.8days) compared to azathioprine is also highly significant (p=2.84×10⁻³).

It is to be understood that the above described examples are forillustration only and that many modifications and variations can be madewithin the scope of the invention.

We claim:
 1. A method of suppressing the immune system of a humancomprising administering to a person requiring same an effective amountof a compound of the formula ##STR30## in which R¹ is a five memberheterocyclic ring system having 2, 3, or 4 nitrogens, or 2 nitrogens and1 sulfur or 1 nitrogen and 1 sulfur, which may be optionally substitutedor unsustituted; R² is hydrogen, lower alkyl or aryl; and R³ ishydrogen, lower alkyl, aryl and nitroaryl, wherein aryl is phenyl ornapthyl.
 2. In the method of claim 1, R² comprising hydrogen, methyl,ethyl or propyl; and R³ is hydrogen, methyl or 4-nitrophenyl.
 3. In themethod of claim 1, said immunosupressive agent having the formula##STR31## wherein R² is hydrogen, methyl, ethyl or propyl and R³ ishydrogen methyl or 4-nitrophenyl, and wherein R¹ is one of thefollowing(i) ##STR32## in which R⁴ is hydrogen or methyl or ethyl orpropyl or phenyl or 1-phenyl OR (ii) ##STR33## in which R⁵ is hydrogenor trifluoromethyl or phenyl or 4-chlorophenyl or 4-methoxyphenyl OR(iii) ##STR34## in which R⁶ is hydrogen or phenyl or chlorophenyl or4-methoxyphenyl OR (iv) ##STR35## in which R¹⁶ is alkyl or aryl OR (v)##STR36## (vi) ##STR37## in which R¹⁷ is alkyl or aryl.
 4. A method ofsuppressing the immune system of a human comprising administering to aperson requiring same an effective amount of a compound of the formula:##STR38## wherein the molecular groups R¹, R² and R³ are defined as oneof the following:(i) R¹ is defined as the following; ##STR39## in whichR⁴ is hydrogen or methyl; R² is ethyl; and R³ is methyl OR R² is methyland R³ is hydrogen; and R⁴ is phenyl or 1-naphthyl OR (ii) R¹ is definedas the following: ##STR40## R²,R³, and R⁵ are one of the followingcombinations: R² is methyl, R³ is 4-nitrophenyl, and R⁵ is hydrogen; orR² is methyl, R³ is hydrogen, and R⁵ is trifluoromethyl; or R² is ethyl,R³ is methyl, and R⁵ is hydrogen; or R² is methyl, R³ is hydrogen, andR⁵ is phenyl; or R² is methyl, R³ is hydrogen, and R⁵ is 4-chlorophenyl;or R² is methyl, R³ is hydrogen, and R⁵ is 4-methoxyphenyl; or R² ismethyl, R³ is hydrogen, and R⁵ is 3-pyridyl OR (iii) R¹ is defined asthe following: ##STR41## in which R² is methyl; and R³ is hydrogen; andR⁶ is phenyl or 4-chlorophenyl or 4-methoxyphenyl OR (iv) R¹ is definedas the following: ##STR42## in which R² is methyl; and R³ is hydrogen;and R¹⁶ is methyl or phenyl OR (v) R¹ is defined as the following:##STR43## OR (vi) R¹ is defined as the following: ##STR44##